We propose that acute lung injury paradigms are likely to be characterized by unique endothelial injury "finger prints" based on the Ca2+ entry pathways they target and the expression pattern of these channel proteins. The consensus of data suggests that members of the canonical subfamily of transient receptor potential (TRP) proteins comprise subunits of store-operated Ca2+ channels and participate in Ca2+ entry-dependent regulation of lung endothelial permeability in extra-alveolar vessels. Our observation that heart failure leads to loss of the permeability response to store depletion but not that to 14,15-epoxyeicosatrienoic acid (14,15-EET), a lipid that promotes Ca2+ entry-dependent acute lung injury only in alveolar septal capillaries, suggests that 14,15-EET targets a distinct channel expressed in septal endothelium. Our preliminary data suggests a novel candidate -TRPV4-a member of the vanilloid subfamily of TRP proteins that is diversely regulated by endocannabinoids, arachidonic acid, EETs, heat, and mechanical perturbation. Our hypothesis is that regulation of TRPV4 channels expressed in alveolar septal endothelium integrates the Ca2+- entry-dependent permeability response to diverse stimuli, including endocannabinoids, EETs, high vascular pressure, hypotonicity, and heat. To address our hypothesis, we have devised 2 specific aims. AIM 1 will determine whether TRPV4 is a common target by which endocannabinoids, EETs, mechanical perturbation, hypotonicity and heat promote Ca2+ influx required for increased endothelial permeability in the alveolar septal compartment of the lung. AIM 2 will reveal whether acute chronic heart failure leads to a selective susceptibility to acute lung injury due to retention of responsiveness to activation of TRPV4 channels but loss of responsiveness to store depletion. We will address these aims using specific measures of permeability and Ca2 + entry in the intact lung endothelium, microscopy of lung and vascular corrosion casts to map spatial heterogeneity in the permeability response, and pharmacological tools to manipulate signaling pathways implicated in gating of TRPV4. The impact of TRPV4 down-regulation in rat lung by siRNA will be compared to outcomes in TRPV4-/ mice, and to that in chronic heart failure where store-operated TRP channels alone appear to be down regulated. This work will provide the first rigorous analysis of compartmentalized TRPV4 channel expression linked to compartmentalized regulation of endothelial permeability in the intact lung.